Development of an SSR marker set for efficient selection for resistance to black spot disease in pear breeding.

Black spot disease, which is caused by Alternaria alternata (Fries) Keissler Japanese pear pathotype, is one of the most harmful diseases in Japanese pear cultivation. Because of the potential harm of fungicides to consumers and the environment, resistant cultivars are desired. In this study, to enable efficient marker-assisted selection in pear breeding, we conducted comprehensive inoculation tests and genotyping with 207 pear cultivars. We identified a marker set (Mdo.chr11.27 and Mdo.chr11.34) suitable for selection for black spot resistance. In most susceptible cultivars, Mdo.chr11.27 amplified a 220-bp band and Mdo.chr11.34 amplified a 259-bp band. The genotype of Mdo.chr11.34 corresponds perfectly to the estimated genotype of Japanese pears susceptible to black spot disease. Using linkage analysis, we identified the positions of the gene for susceptibility to black spot disease in Chinese pear. Mdo.chr11.27 and Mdo.chr11.34 were tightly linked to susceptibility in Chinese pear, and the susceptibility gene was mapped at the top of linkage group 11, similar to that in Japanese pear. This marker set and the accumulation of phenotypic data will enable efficient marker-assisted breeding for black spot resistance in pear breeding.

Identification of candidate genes responsible for the susceptibility of apple (Malus × domestica Borkh.) to Alternaria blotch.

BACKGROUND: The mechanism underlying the interaction between host plant and host-selective toxin (HST)-producing Alternaria alternata during infection is of particular interest for sustainable crop production. Alternaria blotch of apple (Malus × domestica Borkh.) caused by A. alternata apple pathotype is a major disease particularly in East Asia, which is the largest producer of apples globally. A single dominant gene, Alt, controls the susceptibility of the apple cultivar 'Delicious' to Alternaria blotch. In this study, we fine mapped the Alt locus and characterized three potential candidate genes. RESULTS: We used 797 F1 individuals derived from 15 crosses between apple accessions susceptible (Alt/alt) and resistant (alt/alt) to Alternaria blotch to construct physical and genetic maps of the Alt locus located on the top of chromosome 11. Susceptible accessions were derived from 'Delicious.' To fine map the Alt locus, we constructed a BAC library of 'Starking Delicious,' a sport of 'Delicious,' and used graphical genotyping to delimit the Alt locus to a region of 43 kb. Three genes predicted within the candidate Alt region were potentially involved in plant defense response, among which the gene encoding a coiled coil-nucleotide binding-leucine rich repeat (CC-NB-LRR) type disease resistance protein was the most promising. Moreover, a 12-bp insertion was uniquely identified in the 5' untranslated region of the Alt-associated allele of this gene, the presence or absence of which co-segregated with the susceptibility or resistance to A. alternata apple pathotype, respectively, among 43 tested cultivars including old ones and founders of modern apple breeding. CONCLUSION: A disease resistance protein has been suggested as a determinant of susceptibility/resistance to HST-producing A. alternata for the first time. Our finding provides new insight into the mechanism of HST-mediated disease control used by A. alternata against host plants.

Fine mapping of the gene for susceptibility to black spot disease in Japanese pear (Pyrus pyrifolia Nakai).

Black spot disease, which is caused by the Japanese pear pathotype of the filamentous fungus Alternaria alternata (Fries) Keissler, is one of the most harmful diseases in Japanese pear cultivation. We mapped a gene for susceptibility to black spot disease in the Japanese pear (Pyrus pyrifolia Nakai) cultivar 'Kinchaku' (Aki gene) at the top of linkage group 11, similar to the positions of the susceptibility genes Ani in 'Osa Nijisseiki' and Ana in 'Nansui'. Using synteny-based marker enrichment, we developed novel apple SSR markers in the target region. We constructed a fine map of linkage group 11 of 'Kinchaku' and localized the Aki locus within a 1.5-cM genome region between SSR markers Mdo.chr11.28 and Mdo.chr11.34. Marker Mdo.chr11.30 co-segregated with Aki in all 621 F1 plantlets of a 'Housui' × 'Kinchaku' cross. The physical size of the Aki region, which includes three markers (Mdo.chr11.28, Mdo.chr11.30, and Mdo.chr11.34), was estimated to be 250 Kb in the 'Golden Delicious' apple genome and 107 Kb in the 'Dangshansuli' Chinese pear genome. Our results will help to identify the candidate gene for susceptibility to black spot disease in Japanese pear.

Mating-type loci of heterothallic Diaporthe spp.: homologous genes are present in opposite mating-types.

Sexual reproduction of fungi is governed by genes located on the mating-type (MAT) locus. To analyze the MAT locus of the genus Diaporthe (anamorph: Phomopsis), a large genera within the ascomycetous class Sordariomycetes, we cloned and sequenced loci MAT1-1 and MAT1-2 from two heterothallic Diaporthe species, designated as Diaporthe W- and G-types (four isolates in total). The mating-type loci structures of Diaporthe W- and G-types were similar; MAT1-1 isolates had a MAT locus containing three genes, MAT1-1-1, MAT1-1-2 and MAT1-1-3, as was the case with other Sordariomycetes, and in contrast to other Sordariomycetes, MAT1-2 isolates had genes homologous to MAT1-1-2 and MAT1-1-3, in addition to MAT1-2-1. Expression analysis by RT-PCR revealed that all the mating-type genes of Diaporthe W-type were transcriptionally active during vegetative growth. The structure of MAT loci of Diaporthe W- and G-types is distinct from that in other heterothallic filamentous ascomycetes, which have dissimilar gene structure in opposite mating-type loci. This unique structure is informative to discussing the evolutionary history and function of mating-type genes of Sordariomycete fungi.

Ampulla ('takotsubo') cardiomyopathy of both ventricles: evaluation of microcirculation disturbance using 99mTc-tetrofosmin myocardial single photon emission computed tomography and doppler guide wire.

An 84-year-old woman was admitted to hospital with chest pain at rest. An electrocardiogram showed ST-segment elevation in leads II, III, aV(F) and V(2-6), and the 2-dimension echocardiogram showed apical ballooning akinesis and basal hyperkinesis of both ventricles. (99m)Tc-tetrofosmin myocardial single photon emission computed tomography (SPECT) showed severely reduced uptake in the apex. Coronary angiography did not show any organic stenosis, and epicardial coronary spasm was not provoked by the ergonovine loading test. Left ventriculography showed apical ballooning akinesis and basal hyperkinesis, which were also apparent on right ventriculography. The coronary flow velocity pattern showed rapid diastolic acceleration and deceleration times, and the coronary flow reserve measured with a Doppler guide wire was severely decreased. (99m)Tc-tetrofosmin myocardial SPECT showed improvement in the findings after 14 days, and the coronary flow velocity pattern and coronary flow reserve improved after 30 days. Left and right ventriculography both revealed mild improvement in the wall motion. These findings suggested that a microcirculation disturbance caused ampulla ('Takotsubo') cardiomyopathy.

[Efficacy of integrated backscatter imaging by acoustic densitometry for identifying ultrasonic tissue characterization of left ventricular thrombus: two case reports].

Echocardiography demonstrated a high echoic lesion(6 x 5 mm) in the apex of the left ventricle of a 72-year-old man(Case 1) and similar lesions(7 x 6 mm and 4 x 3 mm) in the apex of the left ventricle of a 60-year-old man(Case 2). The amplitude of cyclic variation of integrated backscattering of the high echoic lesions was measured by acoustic densitometry to be lower than that of the myocardium in Case 1, but similar to that of the myocardium in Case 2. Contrast echocardiography detected the perfusion defect of the high echoic lesion in Case 1, but not in Case 2 in the apical four-chamber view. These findings showed that the high echoic lesion indicated thrombus in Case 1, and papillary muscle in Case 2. Measurement of the amplitude of the time intensity curve with contrast echocardiography showed that the amplitude of thrombus was different from that of myocardium. This method is useful for ultrasonic tissue characterization.

[Assessment of microcirculation disturbance with nuclear cardiology in a patient with coronary ectasia: a case report].

A 69-year-old woman presented with dyspnea on exertion. Echocardiography showed dilatation and diffuse hypokinesis of the left ventricle. 99mTc-tetrofosmin myocardial SPECT showed moderately reduced uptake in the anteroseptal wall and the inferior wall on the rest images, but was improved on the ATP loading images. 123I-BMIPP myocardial SPECT showed severely reduced uptake in the anterior wall and the inferior wall. These SPECT findings suggested ischemic heart disease rather than dilated cardiomyopathy. Coronary angiography showed no organic stenosis, but diffuse coronary ectasia was noted in three vessels. Intravascular ultrasound revealed remarkable coronary ectasia, with a maximal diameter of 8.2 mm. Coronary flow velocity as measured by Doppler blood flow guide wire was remarkably reduced. Coronary spasms were not provocated by ergonovine loading test. These findings suggested that microvascular thrombi and disturbance of dilatation caused myocardial ischemia in this patient. We treated the patient with ticlopidine and nicorandil. Following treatment left ventricle wall motion, 99mTc-tetrofosmin and 123I-BMIPP myocardial SPECT findings were improved.

[Marked 99mTc-PYP myocardial accumulation immediately after reperfusion in a patient with acute myocardial infarction].

We reported a case of a 72-year-old man with chest pain. An electrocardiogram showed ST segment elevation in I, II, III, aVL, aVF and V1-6 leads. 99mTc-tetrofosmin myocardial SPECT showed defect in the anterior, septal, apical and inferior walls. Coronary angiography showed 99% stenosis of the proximal right coronary artery and total occlusion of the midsegment of the left anterior descending coronary artery. Therefore, direct PTCA was performed for each lesion to achieve reperfusion. We didnt's see reperfusion injury during PTCA of the left coronary artery. On the other side, we saw severe reperfusion injury, such as slow-flow, arrhythmia and falling blood pressure during PTCA of the right coronary artery. After four hours, 99mTc-PYP myocardial SPECT showed marked uptake in the apical and inferior walls, and mild uptake in the anterior and posterior walls. After three days, severely-reduced uptake of 99mTc-PYP in the apex was noted, and mild uptake in the mid-portion of the anterior wall and the mid-portion of the inferior wall. Though reperfusion injury was seen, three was mild myocardial uptake of 99mTc-PYP in the area of the right coronary artery. On the other side, despite no reperfusion injury, there showed marked uptake during the acute phase and defect during the subacute phase in the area of the left coronary artery. Wall motion of the left ventricle was normal in the area of the right coronary artery and akinesis was seen on the left. These findings suggest that 99mTc-tetrofosmin and 99mTc-PYP myocardial SPECT are useful for visualization of reperfusion injury during the acute phase and for estimation of function during the chronic phase, better even than electrocardiogram or coronary angiography.

[Clinical usefulness of a dual L/N-type Ca2+ channel blocker, cilnidipine, in patients with chronic heart failure: assessment with 123I-MIBG myocardial scintigraphy].

Sympathetic nerve system is activated as a compensatory mechanism in heart failure. However, excessive activation of sympathetic nerve system deteriorates disease state. Sympathetic nerve system can be suppressed with N-type Ca2+ channel blocker. An antihypertensive drug, cilnidipine, is a dual L/N-type Ca2+ channel blocker. We studies usefulness of cilnidipine in treating with chronic heart failure with 123I-MIBG myocardial scintigraphy. We enrolled 24 patients with stable chronic heart failure. Twelve patients were treated with ACE-inhibitors, diuretics and cardiotonics (control group), and the other 12 patients were treated with ACE-inhibitors, diuretics, cardiotonics and cilnidipine (cilnidipine group). We examined blood pressure, heart rate, norepinephrine level, brain natriuretic peptide (BNP) level, cardiothoracic ratio on chest X-ray, ejection fraction of left ventricle on two-dimensional echocardiography, count rate of heart to mediastinum (H/M) and washout rate (WOR) on 123I-MIBG myocardial scintigraphy before and six months after medication. Symptom was improved in 8 patients in the control group and 10 patients in the cilnidipine group after medication. And another parameters were also improved in the both groups after medication. However the degree of change in blood pressure (mmHg) was 21.2 +/- 8.0 in the cilnidipine group and 10.8 +/- 9.1 in the control group, that in heart rate (/min) was 24.1 +/- 6.8 and 16.2 +/- 11.0, that in BNP level (pg/ml) was 65.2 +/- 12.0 and 42.8 +/- 11.1, that in H/M was 0.30 +/- 0.08 and 0.19 +/- 0.09, that in WOR was 19.4 +/- 5.6 and 12.2 +/- 7.0, respectively. And the degree of these changes were larger in the cilnidipine group (p < 0.05). These findings suggested that cilnidipine, a dual L/N-type Ca2+ channel blocker, might be useful in treating with chronic heart failure.

[Serial change of myocardial fatty acid metabolism in a case with severe myocardial ischemia].

A 66-year-old-man was admitted to our hospital because of chest pain on effort in October 1999. The initial images of 123I-BMIPP myocardial SPECT (BM) showed moderately decreased tracer uptake in the apex and the delayed images revealed redistribution in the apex. Coronary angiography revealed 99% stenosis in the proximal portion of the left anterior descending artery. Since sudden onset anaphylactic shock induced by contrast media developed, so percutaneous transluminal coronary angioplasty was not performed. The patient's symptoms were improved with medical treatment. On BM in March 2000, the initial images indicated slightly reduced uptake in the apico-anteroseptal region and the delayed images revealed mildly redistribution in the same area. BM in September 2000, the initial images showed moderately reduced uptake in the apico-anteroseptal region and the delayed images revealed high washout in the same area. The patient's symptoms markedly deferiorrated in March 2001, and BM initial images revealed slightly reduced uptake in the apico-anteroseptal region and the delayed images revealed redistribution in the same area again. During the clinical course, electrocardiography and 99mTc-tetrofosmin myocardial SPECT revealed no marked changes. We consider that 123I-BMIPP myocardial SPECT is useful in estimating myocardial ischemia.

Surgical removal of intra-aortic balloon catheter with fractured nitinol central lumen: a case report.

A 73-year-old woman suffering from septic shock was given circulatory assistance by intra-aortic balloon pumping (IABP). Eleven hours later, pumping stopped abruptly and blood reflux was observed in the intra-aortic balloon catheter (IABC). We removed it and inserted another IABC; 3.5 h later, pumping stopped again and blood reflux was seen. Removal of the IABC was attempted, as the systolic aortic pressure remained above 100 mmHg, but there was resistance during the removal and as a result 7.5cm of the catheter from the tip remained inside the vessel. Fluoroscopy indicated that the metal tube that formed the central lumen in the balloon was fractured, and that its edge had perforated the femoral artery. The balloon was then removed surgically. Fracture of the metal tube and balloon perforation were confirmed in both the damaged IABCs. Postoperative computed tomography and magnetic resonance imaging indicated a highly severe posterior-anterior bend in the patient's aorta. A vessel model similar to the aorta in this case was made and a reproducibility test was conducted; the central lumen fractured within 3 h and under a microscope the profile of the fractured test lumen was similar to the one in the clinical case. These findings suggest that placing a pumping IABC in a bending aorta causes fracture of the central lumen from fatigue failure because the central lumen is under excessive stress.

Assessment of transient left ventricular dilatation on rest and exercise on Tc-99m tetrofosmin myocardial SPECT.

In myocardial perfusion imaging, multiple-vessel involvement of coronary artery disease (CAD) sometimes makes diagnosis difficult. Transient left ventricular (LV) dilatation on Tl-201 myocardial SPECT is a useful finding that enables the clinician to identify patients with multiple-vessel disease. The aim of this study was to confirm the utility of measuring transient LV dilatation for the detection of multiple-vessel CAD in exercise Tc-99m tetrofosmin myocardial SPECT. The participants were 55 CAD patients and 20 controls who underwent Tc-99m tetrofosmin myocardial SPECT exercise and resting imaging. During exercise, 370 MBq (10 mCi) Tc-99m tetrofosmin was injected. Exercise images were obtained 30 minutes after injection. At 210 minutes after injection, 740 MBq (20 mCi) Tc-99m tetrofosmin was administered intravenously. The rest SPECT images were acquired 30 minutes later. Thirty-six radii at every 10 degrees were generated from the center of short-axis images. An area surrounded by 36 maximal points of the myocardial Tc-99m tetrofosmin counts on each radius was calculated for exercise and rest images. The area surrounded by the 36 maximal points in the same slice of the exercise and rest images was assigned the variables A (Ex) and A(R), respectively. The transient dilatation index (TDI) of the left ventricle was calculated using the formula mean A (Ex)/A(R) in the apical, middle, and basal myocardial short-axis images. In the controls, the TDI was 0.970 +/- 0.021. In patients with CAD, the TDIs of one-vessel disease, two-vessel disease, and three-vessel disease were 1.034 +/- 0.032, 1.093 +/- 0.046, and 1.131 +/- 0.076, respectively. The TDIs were significantly greater in patients who had more occluded coronary arteries (P < 0.01). If the mean + 2SD of the TDI (1.012) in controls were assumed to be the normal upper limit, the sensitivity, specificity, and accuracy of this method in detecting two-vessel or three-vessel disease would be 91.4%, 76.9%, and 84%, respectively. The TDI is a useful index for evaluating subendocardial ischemia non-invasively and detecting multiple-vessel disease clinically.

Increasing myocardial 123I-BMIPP uptake in non-ischemic area in a patient with acute myocardial ischemia.

The subject was a 65-year-old woman with chest pain. An electrocardiogram revealed T-wave-inversion in leads III, aVF, V1-V5. 99mTc-tetrofosmin myocardial SPECT showed mildly reduced uptake in the anteroseptal wall and the apex. These findings suggested acute myocardial ischemia. Coronary angiography did not show any stenotic lesions, but diffuse coronary ectasia was noted in three vessels. Coronary flow velocity was remarkably reduced on coronary angiography. Epicardial coronary spasm was not provoked by ergonovine loading test. Left ventriculography showed diffuse hypokinesis. 123I-BMIPP myocardial SPECT showed mildly reduced uptake in the anteroseptal wall and the apex on the early images. But 4-hour delayed images showed an increase of 8% in myocardial 123I-BMIPP uptake. We treated this patient with ticlopidine and nicorandil. After drug therapy her symptoms and left ventriculography improved. 123I-BMIPP myocardial SPECT findings on the early images improved, whereas delayed images showed a decrease of 28% in myocardial 123I-BMIPP uptake after two weeks and 36% after four weeks. These dynamic changes in 123I-BMIPP findings might be a reflection of myocardial fatty acid metabolism in patients with acute myocardial ischemia. Delayed 123I-BMIPP myocardial SPECT images are useful for the assessment of fatty acid metabolism.

[A case of "Takotsubo" cardiomyopathy observed with myocardial scintigraphy from the acute phase].

A 57-year-old woman was emergently admitted to our hospital because of chest oppression. On examination, blood pressure was 174/96 mmHg, pulse rate was 90/min and coarse crackle and third heart sound were audible. On laboratory data, the levels of LDH and CPK-MB were mildly elevated. Electrocardiogram (ECG) demonstrated elevation of the ST segment in leads I, aVL and V2-V5. 99mTc-tetrofosmin myocardial SPECT (TF) showed severely reduced uptake in the apex and anterior wall. Emergent coronary angiography (CAG) did not show any stenotic lesion. Left ventriculography (LVG), however, demonstrated akinesis of the apex, anterior, and inferior walls, and basal hyperkinesis. On the second hospital day, ECG demonstrated inverted T wave in leads I, aVL and V2-V5. 123I-BMIPP myocardial SPECT (BMIPP) and 123I-MIBG myocardial SPECT (MIBG) were performed on the second and fourth hospital days, respectively. These cardiac images showed severely reduced uptake equally. TF, BMIPP, and MIBG were re-examined on the eighth, tenth, and twelfth hospital days, respectively. MIBG, BMIPP, and TF showed reduced uptake in order of severity. On the fourteenth hospital day, CAG and LVG were re-examined. Coronary vasospasm provocation test was negative using ergonovine and acetylcholine, and LVG did not demonstrated any sign of asynergy. We considered that this case was "Takotsubo" cardiomyopathy and might be caused by microvascular spasm.